Wild-type Fibroblasts Research Articles

Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†.

Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the β3 subunit of voltage-gated calcium channels (Cavβ3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cavβ3-deficient mice (Cavβ3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cavβ3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cavβ3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cavβ3-deficient mice or cells might also be applicable for specific molecules other than Cavβ3.

Focal adhesion kinase regulates tractional collagen remodeling, matrix metalloproteinase expression, and collagen structure, which in turn affects matrix-induced signaling.

Focal adhesion kinase (FAK) is critical for collagen expression but its regulation of collagen remodeling is not defined. We examined the role of FAK in the degradation and reorganization of fibrillar collagen. Compared with wild-type (WT) mouse embryonic fibroblasts, FAK null (FAK-/- ) fibroblasts generated twofold (p < .0001) higher levels of ¾ collagen I fragment and expressed up to fivefold more membrane-type matrix metalloproteinase (MMP). When plated on stiff collagen substrates, compared with WT, FAK-/- cells were smaller (threefold reduced cell surface area; p < .0001) and produced fivefold fewer cell extensions (p < .0001) that were 40% shorter (p < .001). When cultured on soft collagen gels (stiffness of ~100 Pa) for 6-48 hr, cell spreading and cell extension formation were reduced by greater than twofold (p < .05 and p < .0001, respectively) while collagen compaction and alignment were reduced by approximately 30% (p < .0001) in FAK-/- cells. Similar results were found after treatment with PF573228, a FAK inhibitor. Reconstitution of FAK-/- cells with FAK mutants showed that compared with WT, cell extension formation was reduced twofold (p < .0001) in the absence of the kinase domain and sixfold (p < .0001) with a Y397F mutant. Enhanced collagen degradation was exhibited by the mutants (~threefold increase; p < .0001 of ¾ collagen fragments without kinase domain or Y397F mutant; p < .01). Compared with FAK+/+ cells, matrices produced by FAK-/- cells generated higher levels of β1 integrin activation (p < 0.05), extracellular-signal-regulated kinase (ERK) phosphorylation, and production of ¾ collagen I fragment by human gingival fibroblasts. Collectively these data indicate that (a) the kinase activity of FAK enhances collagen remodeling by tractional forces but inhibits collagen degradation by MMPs; (b) FAK influences the biological activity of fibroblast-secreted extracellular matrices, which in turn impacts β1 integrin and ERK signaling, and collagen degradation.

The role of vimentin in directional migration of rat fibroblasts.

Cell migration is one of the most important processes in which the cytoskeleton plays a main role. The cytoskeleton network is formed by tubulin microtubules, actin filaments, and intermediate filaments (IFs). While the structure and functions of the two aforementioned proteins have been extensively investigated during the last decades, vimentin IFs structure and their role in cell migration and adhesion remain unclear. Here, we investigated polarity determination in rat fibroblasts with either a knocked out vim gene or with a mutation that blocks filament formation on the stage of unit-length filaments (ULFs). Structured illumination microscopy has demonstrated the difference in the morphology of IFs in wild-type fibroblasts and of ULFs in mutant fibroblasts. We have developed an approach to measure cell stiffness separately on the trailing and leading edges using atomic force microscopy. Young's modulus values on the leading and trailing edge of migrating rat fibroblasts differ approximately by two times, being larger on the leading edge. The knockout of the vim gene leads to having comparable values of Young's moduli on both edges. Vimentin-null cells change the direction of migration more frequently than those expressing wild-type or mutated vimentin. Our results have shown the principle role of vimentin, not only in the form of IFs, but also as ULFs, in the determination of the polarity and the directionality of fibroblast migration.

AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.—Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

Deletion of Calcineurin Promotes a Protumorigenic Fibroblast Phenotype.

Fibroblast activation is a crucial step in tumor growth and metastatic progression. Activated fibroblasts remodel the extracellular matrix (ECM) in primary tumor and metastatic microenvironments, exerting both pro- and antitumorigenic effects. However, the intrinsic mechanisms that regulate the activation of fibroblasts are not well-defined. The signaling axis comprising the calcium-activated Ser/Thr phosphatase calcineurin (CN), and its downstream target nuclear factor of activated T cells, has been implicated in endothelial (EC) and immune cell activation, but its role in fibroblasts is not known. Here, we demonstrate that deletion of CN in fibroblasts in vitro altered fibroblast morphology and function consistent with an activated phenotype relative to wild-type fibroblasts. CN-null fibroblasts had a greater migratory capacity, increased collagen secretion and remodeling, and promoted more robust EC activation in vitro. ECM generated by CN-null fibroblasts contained more collagen with greater alignment of fibrillar collagen compared with wild-type fibroblast-derived matrix. These differences in matrix composition and organization imposed distinct changes in morphology and cytoskeletal architecture of both fibroblasts and tumor cells. Consistent with this in vitro phenotype, mice with stromal CN deletion had a greater incidence and larger lung metastases. Our data suggest that CN signaling contributes to the maintenance of fibroblast homeostasis and that loss of CN is sufficient to promote fibroblast activation. SIGNIFICANCE: Calcineurin signaling is a key pathway underlying fibroblast homeostasis that could be targeted to potentially prevent fibroblast activation in distant metastatic sites.

HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress

High mobility group box-1 (HMGB1) is involved in various diseases and is associated with the resistance of many types of human cancers to chemotherapy; however, its role in cancer metastasis remains unexplored. This study examined the HMGB1 status of both highly and poorly metastatic cancer cells in response to genotoxic stress. The weakly and highly metastatic mouse melanoma cell lines (B16 vs. B16-F10), human melanoma cell lines (SK-MEL-28 vs. SK-MEL-24), colon cancer cell lines (DLD-1 vs. LS174T), and wild-type (WT) vs. HMGB1 knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (Dox) and camptothecin (CPT), and then cellular morphology, senescence-associated β-galactosidase staining, lactate dehydrogenase release, and caspase-3 activation were used to assess cell fate. To investigate the role of HMGB1 in p21 expression, HMGB1 and p21 expressions were examined by Western blotting, and the HMGB1-mediated p21 promoter luciferase assay was performed after small interfering RNA or overexpression of HMGB1 prior to Dox treatment. Although highly metastatic mouse melanoma B16-F10 cells preferred senescence, with persistent HMGB1 expression, poorly metastatic B16 cells entered apoptosis, with decreasing HMGB1 levels via cleavage under Dox treatment. Similarly, more metastatic human melanoma SK-MEL-24 and human colon cancer LS174T cells underwent senescence, whereas fewer metastatic melanoma SK-MEL-28 and DLD-1 cells exhibited apoptosis under Dox stimulation. In senescent B16-F10, SK-MEL-24, and LS174T cells treated with Dox, p21 levels were increased by persistent HMGB1 expression. Furthermore, HMGB1 depletion caused a senescence-apoptosis shift with p21 down-regulation in B16-F10 cells, and HMGB1 overexpression switched from apoptosis to senescence concomitantly with increased p21 expression in B16 cells after Dox treatment. The same effects were observed in both cell pairs of mouse melanoma and human colon cancer cells treated with CPT, another genotoxic stressor. Indeed, although WT MEF entered senescence accompanied by p21 increase, HMGB1 KO underwent apoptosis with p21 decrease by Dox treatment. In our cell model system, we demonstrated that highly metastatic cancer cells preferentially enter senescence, whereas apoptosis predominates in weakly metastatic cancer cells under genotoxic stress, which depends on the presence or absence of HMGB1, suggesting that the HMGB1-p21 axis is required for genotoxic stress-induced senescence. These findings suggest that HMGB1 modulation of cancers with different metastatic status could be a strategy for selectively enforcing tumor suppression.-Lee, J.-J., Park, I. H., Rhee, W. J., Kim, H. S., Shin, J.-S. HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

Abstract 5164: Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression

Abstract Juxtacrine signaling with the stromal compartment is important to the development of castrate resistance prostate cancer. We found that Notch signaling is upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs resulted in decreased Wnt signaling and decrease in expression of Notch pathway genes. Interestingly, androgen receptor antagonism resulted in increased Notch signaling and the upregulation of downstream Wnts. Further, generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling that are derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts of RBPJ-knockout mice that have a loss of Notch signaling demonstrated diminished tumorigenesis, compared with xenografts with the wild type fibroblasts. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells caused increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together our results suggest that there is a stromal-derived feed-back - feed-forward signaling loop involving Notch and Wnt ligands that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can potentially limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer. Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Rajeev Mishra, Leland Chung, Li Xin, Neil Bhowmick. Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5164.

Abstract SY20-02: The SIRT3-MnSOD-Ac axis in carcinogenesis and tumor resistance

Abstract ABSTRACT The molecular mechanism by which acetylation regulates manganese superoxide dismutase (MnSOD) activity, and its oncogenic and/or physiological significance, is unclear. Here we show that expression of the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) transformed wild-type primary mouse embryonic fibroblasts (pMEFs) when co-infected with either c-Myc or Ras, as measured by in vitro transformation assays and xenograft formation. Immortalized MnSOD-/- mouse embryonic fibroblasts (MEFs) infected with lenti-MnSODK68Q also exhibited an in vitro transformation permissive phenotype. In addition, MCF7 and T47D cells expressing MnSODK68Q demonstrated increased in vitro and xenograft oncogenic properties. These findings collectively identify MnSODK68Q as a tumor promoter. MCF7, T47D, and MnSOD-/- cells expressing MnSODK68Q displayed a monomeric form of MnSOD, distinct from the well-established homotetramer, and MnSOD-K68-Ac functions as a peroxidase, determined using a bacterial system for site-specific incorporation of N-(ε)-acetyl-l-lysine. Finally, tamoxifen resistant MCF7 and T47D cells, and a subgroup of human breast malignancies, displayed a MnSOD-K68-Ac signature. Citation Format: David R. Gius, Yueming Zhu. The SIRT3-MnSOD-Ac axis in carcinogenesis and tumor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY20-02.

A porous collagen-carboxymethyl cellulose/hydroxyapatite composite for bone tissue engineering by bi-molecular template method

Inspired by the mechanism of bone formation, a porous collagen-carboxymethyl cellulose/hydroxyapatite (Col-CMC/HA) composite was designed and fabricated using a biomimetic template of Col and CMC protein-polysaccharide bi-molecules. The morphology, composition and physical properties of Col-CMC/HA composites were characterized systematically. It was found that the nano-HA homogenously distributed on the surface of Col-CMC bi-templates while the composite presented 3D porous structure with pore size from 100 μm to 300 μm. The porosities of composites were located at the range of 71%–85%. Besides, the compressive strength of composites was highly depended on the ratio of Col to CMC in the organic template. The optimized composite in respect to physical properties showed a compressive strength as high as 7.06 MPa, quite close to that of natural bone. The high relative growth rate of wild-type mouse embryonic fibroblasts cells was found for the composite, indicating a good biocompatibility. The organic-inorganic composite also behaved good in collagenase resistance and could be biodegraded in 8 weeks, with about 50% of initial weight left at the ratio of Col to CMC of 1:9. The results demonstrated that the Col-CMC/HA composite by bi-molecular template method was a rational and safe method to prepare biomaterials with tunable properties.

Endocrine aryl hydrocarbon receptor signaling is induced by moderate cutaneous exposure to ultraviolet light

Links between solar UV exposure and immunity date back to the ancient Greeks with the development of heliotherapy. Skin contains several UV-sensitive chromophores and exposure to sunlight can produce molecules, such as vitamin D3, that act in an endocrine manner. We investigated the role of the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-regulated transcription factor activated by numerous planar compounds of endogenous, dietary or environmental origin. 15- to 30-minute exposure of cells to a minimal erythemal dose of UVB irradiation in vitro induced translocation of the AHR to the nucleus, rapidly inducing site-specific DNA binding and target gene regulation. Importantly, ex vivo studies with Ahr wild-type or null fibroblasts showed that serum from mice whose skin was exposed to a 15 min UVB dose, but not control serum, contained agonist activity within 30 min of UV irradiation, inducing AHR-dependent gene expression. Moreover, a 15-min cutaneous UVB exposure induced AHR site-specific DNA binding and target gene regulation in vivo within 3–6 hr post-irradiation in blood and in peripheral tissues, including intestine. These results show that cutaneous exposure of mice to a single minimal erythemic dose of UVB induces rapid AHR signaling in multiple peripheral organs, providing compelling evidence that moderate sun exposure can exert endocrine control of immunity through the AHR.

MMP14-Containing Exosomes Cleave VEGFR1 and Promote VEGFA-Induced Migration and Proliferation of Vascular Endothelial Cells.

PurposeInvestigate the impact matrix metalloproteinase 14 (MMP14) delivered via exosomes produced by corneal fibroblasts on vascular endothelial growth factor receptor 1 (VEGFR1) cleavage on endothelial cells, and other key processes of angiogenesis.MethodsProteolysis of VEGFR1 and R2 by the catalytic domain of MMP14 was investigated via immunocytochemistry with anti-VEGFR1, anti-VEGFR2, and anti-MMP14 antibodies. Exosomes were isolated via precipitation and serial ultracentrifugation from wild-type (WT) and MMP14 exon4-deficient corneal fibroblasts. Transmission electron microscopy and nanotracking analysis were used to characterize the isolated exosomes. The presence of MMP14 in exosomes from WT fibroblasts was confirmed by Western blotting. VEGFR1 cleavage upon treatment with WT-derived exosomes, Δexon4-derived exosomes, or the pan-MMP inhibitor GM60001 was examined via in vitro proteolysis analysis using recombinant mouse (rm) VEGFR1/R2. Endothelial cell migration and proliferation were investigated using a Boyden chamber assay and BrdU incorporation, respectively.ResultsWT-derived exosomes specifically cleaved rmVEGFR1 in vitro, whereas Δexon4-derived exosomes did not. Treatment with the pan-MMP inhibitor GM6001 effectively inhibited VEGFR1 cleavage by WT-derived exosomes, confirming the role of MMP14 in this cleavage. WT-derived exosomes induced greater endothelial cell migration (P < 0.01) and proliferation (P < 0.5) compared to Δexon4-derived exosomes.ConclusionsMMP14-containing exosomes may be involved in the regulation of corneal neovascularization through degradation of VEGFR1 and VEGFA-induced endothelial cell proliferation and migration.

SIRT7 regulates the nuclear export of NF-κB p65 by deacetylating Ran

Sirtuin 7 (SIRT7) is an NAD+-dependent lysine deacetylase that regulates diverse biological processes. We recently observed that SIRT7 deficiency suppresses the nuclear accumulation of p65, which is a component of nuclear factor kappa B. However, the underlying molecular mechanism remains elusive. In this study, we demonstrated that SIRT7 interacts with a small GTPase, Ras-related nuclear antigen (Ran), and deacetylates Ran at K37. The nuclear export of p65 was facilitated in SIRT7-deficient fibroblast cells, while the nuclear export was inhibited in SIRT7-deficient cells expressing K37R-Ran (deacetylation-mimicking mutant). Additionally, the nuclear export of p65 in wild-type fibroblast cells was promoted by K37Q-Ran (acetylation-mimicking mutant). K37Q-Ran exhibited an increased ability to bind to chromosome region maintenance 1 (CRM1), which is a major nuclear receptor that mediates the export of cargo proteins, and enhanced the binding between p65 and CRM1. These data suggest that SIRT7 is a lysine deacetylase that targets the K37 residue of Ran to suppress the nuclear export of p65.

Proteomics Profiling of Cyclosporine A Treatment of Calcineurin Isoform‐Specific Renal Fibroblasts

Background: Metalloproteinases (MMPs) produced by renal fibroblasts have been implicated in the initiation and progression of calcineurin inhibitor‐induced nephrotoxicity. Our lab demonstrated that loss of the calcineurin A alpha (CnAα) isoform (or presence of the calcineurin A beta (CnAβ) isoform) is associated with significantly enhanced CsA‐induced MMP‐9 secretion vs. in wild‐type renal fibroblasts. Understanding the role of calcineurin A isoforms in the regulation of the homeostasis of extracellular matrix (ECM) degradation in the kidney after cyclosporine A (CsA) treatment needs to be further investigated. Proteomic studies in cell cultures, animal models and human tissues have contributed to our current knowledge and understanding of the pathophysiological mechanisms involved in renal fibrosis biology. Comparative proteomics characterization of our calcineurin A isoform‐specific renal fibroblasts can provide new insights into MMP‐9 and other protein biomarkers associated with mechanisms underlying the differential expression of CsA‐induced calcineurin isoform‐specific proteins associated with renal fibrosis.ObjectiveThis study uses our established CnA isoform‐specific cell lines and proteomics to highlight the changes in secreted proteins associated with CsA‐induced kidney fibrosis and whether those changes are calcineurin A isoform‐specific.Methods and ResultsWild‐type (WT) and CnA α knockout (CnAα−/−) kidney fibroblast cell lines were treated with CsA at 10 ng/ml for 24 and 48 hrs in serum‐free DMEM media. To characterize the CsA‐induced protein profiles of our renal fibroblast cells, supernatants were collected and analyzed using a Thermo Q‐Exactive HF‐X Orbitrap mass spectrometer. Our results revealed that after CsA treatment for 24 hrs, significantly higher protein levels of MMP‐2 and MMP‐9 (3‐ and 6‐fold, respectively) were secreted from CnAα−/− cells compared to WT cells. At 48 hrs, the levels of MMP‐9 increased even higher (6.5 to 11‐fold) in CnAα−/− cells vs. WT cells. However, the levels of MMP‐2 in CnAα−/− cells decreased at 48 hrs. Interestingly, the proteomic profile of top proteins expressed in WT vs. CnAα−/− were differentially regulated. At 24 hrs, the top secreted proteins from WT fibroblasts were shown to be involved in regulating T‐cell development and function. At 24 hrs in CnAα−/− cells, the top secreted proteins included biomarkers of structural elements of ECM (i.e. Col6, FN, Fbn1), metalloproteinases (i.e. MMP‐2, MMP‐9), unfolded protein response pathway and ER‐stress response proteins (i.e. GRP78/BIP, ERP57, CALR), and oxidative stress pathway proteins (i.e. HYOU1, HSP7070, HSP90, PRDX1). By 48 hrs, these CsA‐induced pro‐fibrotic proteins were significantly upregulated in CnAα−/− cells (Range: 3‐ to 242‐fold).ConclusionsThe proteomics data presented strongly confirms the differences in secreted MMP‐9 and MMP‐2 levels we previously demonstrated between our calcineurin A isoform‐specific cell lines. Like other proteomics approaches to study renal fibrosis, we observed similar upregulated pro‐fibrotic/fibrotic protein profiles. Our results also demonstrated that the upregulation of other pro‐fibrotic proteins are also calcineurin A isoform specific.Support or Funding InformationFunded by NIH Grant 1R15GM113120‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of Function in Dopamine Receptor‐3 (D3R) Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation In Vitro

Dopamine is a neurotransmitter heavily involved in neural pathways regulating the reward response, body movement, mood, and cognitive function. As such, much of the research on dopamine and its five receptor subtypes has been conducted to observe the in vivo function of each receptor subtype in neurological disorders. However, recent studies by colleagues from our institution have suggested that D3Rs may play a role in cardiac‐related aging, as 2‐month old D3 receptor knock‐out (D3KO) mice show age‐related changes in cardiac function similar to 2‐year old wild‐type (WT) mice. Thus, it would be interesting to understand the role of D3R expression in LV cardiac fibroblast and its function. We isolated, cultured and characterized cardiac fibroblasts from the left ventricles (LV) of WT (23‐weeks old) and D3KO (5 and 31‐week‐old) mice. Immunohistochemistry was performed to determine the expression of D3R in LV cardiac fibroblasts, and cell culture experiments were performed to examine possible changes in WT and D3KO cell migration and proliferation. Proliferation was measured via total cell count over time points of 6, 12, 24, and 36 hours, while migration in response to scratch wound was examined as percent change in area at 3, 6, 12, 24 hours, and every 12 hours thereafter up to 86 hours. Staining with antibodies for D3R successfully showed expression of this receptor in WT fibroblasts, while cell culture experiments showed reduced proliferation and migration of D3KO fibroblasts compared to WT. Migration of D3KO fibroblasts from 5‐week old mice were significantly different (*p&lt;0.05) in response to an artificial wound when compared to 23‐week‐old mice. Additionally, upon isolation of LV cardiac fibroblasts from 5‐week‐old and 31‐week‐old D3KO and 23‐week‐old WT mice, cell volume from D3KO fibroblast isolation was markedly reduced compared to WT isolation under identical conditions (5‐fold and 10‐fold decrease in 5‐week and 31‐week D3KO, respectively). This data suggests that D3Rs play a role in LV cardiac fibroblast migration and proliferation and support previous observations of age‐related changes in cardiac function of D3KO mice.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model.

Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler's disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.

NADPH oxidase-mediated induction of reactive oxygen species and extracellular matrix deposition by insulin-like growth factor binding protein-5.

Insulin-like growth factor binding protein-5 (IGFBP-5) induces production of the extracellular matrix (ECM) components collagen and fibronectin both in vitro and in vivo and is overexpressed in patients with fibrosing lung diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). However, the mechanism by which IGFBP-5 exerts its fibrotic effect is incompletely understood. Recent reports have shown a substantial role of reactive oxygen species (ROS) in fibrosis; thus we hypothesized that IGFBP-5 induces production of ROS to mediate the profibrotic process. In vitro analyses revealed that ROS production was induced by recombinant and adenoviral vector-mediated IGFBP-5 (AdBP5) in a dose- and time-dependent manner, regulated through MEK/ERK and JNK signaling, and primarily mediated by NADPH oxidase (Nox). Silencing IGFBP-5 in SSc and IPF fibroblasts reduced ROS production. The antioxidants diphenyleneiodonium and N-acetylcysteine blocked IGFBP-5-stimulated ECM production in normal, SSc, and IPF human primary lung fibroblasts. In murine fibroblasts lacking critical components of the Nox machinery, AdBP5-stimulated ROS production and fibronectin expression were reduced compared with wild-type fibroblasts. IGFBP-5 stimulated transcriptional expression of Nox3 in human fibroblasts while selective knockdown of Nox3 reduced ROS production by IGFBP-5. Thus IGFBP-5 mediates fibrosis through production of ROS in a Nox-dependent manner.

Optimisation of seeding of wild-type and FOP fibroblasts for an alkaline phosphatase assay using calcein AM staining

Optimisation of seeding of wild-type and FOP fibroblasts for an alkaline phosphatase assay using calcein AM staining

Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

Mouse mast cell protease-4 (mMCP4) is a chymase that has been implicated in cardiovascular diseases, including myocardial infarction (MI). This study tested a direct role of mMCP4 in mouse post-MI cardiac dysfunction and myocardial remodeling. Immunoblot and immunofluorescent double staining demonstrated mMCP4 expression in cardiomyocytes from the infarct zone from mouse heart at 28 day post-MI. At this time point, mMCP4-deficient Mcpt4−/− mice showed no difference in survival from wild-type (WT) control mice, yet demonstrated smaller infarct size, improved cardiac functions, reduced macrophage content but increased T-cell accumulation in the infarct region compared with those of WT littermates. mMCP4-deficiency also reduced cardiomyocyte apoptosis and expression of TGF-β1, p-Smad2, and p-Smad3 in the infarct region, but did not affect collagen deposition or α-smooth muscle actin expression in the same area. Gelatin gel zymography and immunoblot analysis revealed reduced activities of matrix metalloproteinases and expression of cysteinyl cathepsins in the myocardium, macrophages, and T cells from Mcpt4−/− mice. Immunoblot analysis also found reduced p-Smad2 and p-Smad3 in the myocardium from Mcpt4−/− mice, yet fibroblasts from Mcpt4−/− mice showed comparable levels of p-Smad2 and p-Smad3 to those of WT fibroblasts. Flow cytometry, immunoblot analysis, and immunofluorescent staining demonstrated that mMCP4-deficiency reduced the expression of proapoptotic cathepsins in cardiomyocytes and protected cardiomyocytes from H2O2-induced apoptosis. This study established a role of mMCP4 in mouse post-MI dysfunction by regulating myocardial protease expression and cardiomyocyte death without significant impact on myocardial fibrosis or survival post-MI in mice.

3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts withHuman Prostate Cancer Epithelial Cells.

Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression. This was established using transgenic mouse models. Paracrine interactions between fibroblasts and epithelial cells were further interrogated using isolated 2D cell culture systems, but 3D culture systems currently being developed can better mimic reciprocal interactions potentially found in the native tissue. To understand paracrine and juxtacrine signaling among fibroblasts and epithelia, 3D co-cultures with species differences allows for further subsequent analysis of the cultures. The use of mouse and human cells, for example, in one system allows for species-specific FACS or quantitative PCR analysis. This protocol describes the use of a 3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells.